Ab16048

The purified sEVs and A549 cells were lysed with 1 × RIPA buffer and cocktails and centrifuged at 13,000×g for 15 min to obtain the supernatant. Protein concentrations were detected with BCA Protein Assay according to the manufacturer’s protocols. sEVs markers CD9 (Abcam, ab92726), CD63 (Abcam, ab193349), TSG101 (Abcam, ab83), and sEVs non-markers calnexin (Abcam, ab22595), GM130 (Abcam, ab52649), Lamin B1 (Abcam, ab16048), and GRP94 (Proteintech, 14700-1-AP) were measured to validate the expressions of sEVs as described in prior studies^{52 (link),53 (link)}.

Cell pellets were lysed in lysis buffer (0.1 M Tris-HCl (pH 7.5), 10% glycerol, and 1% sodium dodecyl sulfate (SDS)), boiled for 5 min, and then centrifuged for 10 min at 15,000 rpm. All protein concentrations were determined using BCA Protein Assay Reagent (Pierce). Each cell lysate was electrophoresed using SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore). After blocking with 5% skim milk (Megmilk) or 5% bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween 20 (TBST), the membrane was treated with primary antibodies against Ras (1:1000, Oncogene Research Products, #OP41), lamin-B1 (1:1000, Abcam, #ab16048), GAPDH (1:10,000, Proteintech, #60004-1), and phospho-Met (Tyr1234/1235) (D26) (1:1000, Cell Signaling Technology, #3077) overnight at 4 °C in a blocking buffer. Membranes were then washed thrice in TBST and incubated with enhanced chemiluminescence (ECL) anti-mouse IgG, horseradish peroxidase-linked whole antibody (GE Healthcare, #NA931V) or ECL anti-rabbit IgG, and horseradish peroxidase-linked whole antibody (GE Healthcare, #NA934V) for 1 h at room temperature. After washing the membrane thrice with TBST, the signal was resolved using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and imaging was performed using a FUSION imaging system (Vilber-Lourmat).